The habenula is a paired nucleus residing in the dorsal thalamus. It consists of medial and lateral subnuclei (MHb, LHb), which connect to distinct targets in the ventral midbrain. Lesion studies of the entire habenula in animals have implicated this area in diverse functions, including circadian rhythms and sleep, stress responses, intracranial self-stimulation reward, and the behavioral effects of nicotine and other drugs of abuse. In humans, a few preliminary clinical studies have linked habenula reward pathways to possible mechanisms of depression, but the function of this nucleus remains largely unclear. Recent work has shown that some LHb neurons inhibit dopamine (DA) signaling in the ventral tegmental area (VTA), and thus mediate a negative reward signal. In contrast, the MHb projects almost exclusively to the interpeduncular nucleus (IP), adjacent to the VTA, which projects in turn to the raphe and dorsal tegmental areas. The MHb has dorsal (dMHb) and ventral (vMHb) subnuclei, which make specific connections to the lateral and medial IP, respectively. Prior studies have not distinguished the roles of these subnuclei, yet there is littl reason to believe they have the same function. Our new optogenetic data show that, in contrast to the LHb, stimulation of the dMHb generates a positive reward signal, and we hypothesize that this is mediated via a MHb-IP pathway to the brainstem. Here we will use Cre-drivers specific for the dMHb and vMHb, combined with transgenic mice that allow inducible expression of the optogenetic proteins Channelrhodopsin and Halorhodopsin, to activate and silence MHb neurons in order to examine the functional link from the MHb to GABA neurons in the IP. Genetically targeted approaches will also be used to examine the IP connection to 5HT neurons in the raphe, using neuroanatomical, electrophysiological, and behavioral readouts. Aim 1. We will use Cre-mediated transgenic, viral, and conventional tract-tracing to determine the anatomical connectivity of pathways from the habenula, via the IP, to hindbrain centers mediating reward and fear responses. We will assign neurotransmitter phenotypes to the neurons in these pathways to determine whether they transmit excitatory or inhibitory signals. Aim 2. We will test the physiological connections between the MHb-IP and the IP-raphe in brain slice preparations, using optogenetic activation of specific presynaptic neurons combined with intracellular recording of postsynaptic neurons, and identification of the recorded neurons with transgenic markers. Aim 3. We will use optogenetics to activate and silence the dMHb and vMHb in behavioral models of reward and depression, including open field locomotion, intracranial self-stimulation, real-time place preference/aversion, conditioned place preference/aversion, and learned helplessness. At the conclusion of these experiments we will better understand the specific habenula components and their downstream pathways, and the potential role of the habenula in mood disorders.